Antibody drug conjugates

ABSTRACT

The present invention pertains to an antibody-drug conjugate comprising an antibody, ferric iron, and at least one drug molecule, and to a pharmaceutical composition comprising the antibody-drug conjugate. The invention further relates to the use of the antibody-drug conjugates in the treatment of diseases, e.g. cancer.

This application corresponds to the U.S. national phase of InternationalApplication No. PCT/EP2017/079869, filed Nov. 21, 2017, which, in turn,claims priority to European Patent Application No. 16.200071.5 filedNov. 22, 2016, the contents of which are incorporated by referenceherein in their entirety.

FIELD OF THE INVENTION

The present invention relates to the use of antibody-drug conjugates inthe treatment of diseases such as cancer.

BACKGROUND

Traditional cancer chemotherapy is often accompanied by systemictoxicity to the patient. Targeted therapy approaches seek tospecifically interfere with molecular targets and pathways that areimportant for the proliferation of cancer cells. These targets arepreferentially expressed either intracellularly or on the surface oftumor cells. Thus, targeted therapy offers the potential to generateagents that will be selectively cytotoxic to tumor cells, coupled withlower toxicity to the host, resulting in a larger therapeutic index. Oneapproach was the development of inhibitors of tyrosine kinases that aremisregulated and/or overexpressed in cancer cells. Another targetedtherapy approach is the use of small molecules that selectively bind tothe surface of tumor cells to deliver a cytotoxic compound (e.g. afolate-vinca alkaloid conjugates).

Monoclonal antibodies against antigens on cancer cells offer analternative tumor-selective treatment approach. However, many monoclonalantibodies are not sufficiently potent to be therapeutically active ontheir own. Antibodydrug conjugates (ADCs) use antibodies to deliver apotent cytotoxic compound selectively to tumor cells, thus improving thetherapeutic index of chemotherapeutic agents. Two of the most prominentexamples are the approved drugs ado-trastuzumab emtansine (Kadcyla®) andbrentuximab vedotin (Adcetris®).

Current ADCs, however, often suffer from insufficient potency of the ADCas compared to the parent free drug. This may be due to insufficientcleavage from the antibody after the ADC is being taken up by the cancercell and processed in the endosome/lysosome. Current ADCs often need tobe linked to the antibody using a specialized linker that cannot or onlypartially be cleaved off during intracellular metabolism.

Another drawback of certain ADCs is their poor stability in the blood.Labile linkers release the drug too early, which results in loss ofactivity of the ADC and increased negative side-effects. On the otherhand, highly stable linkers hamper efficient release once the ADCreaches the endosome.

In addition, it is generally difficult to achieve a sufficiently highintracellular concentration of cytotoxic compound, as the number ofantigens on a cancer cell is typically ≤10⁵. However, the intracellularconcentration of cytotoxic compound should preferably be significantlyhigher than 10⁵.

WO 2013/103707 A1 discloses various ADCs comprising a platinum cation.

Thus, there is a need for improved antibody drug conjugates overcomingone or more of the disadvantages of existing therapies.

SUMMARY OF THE INVENTION

The inventors of this application found that ADCs in which the drug islinked to the antibody via ferric iron are stable and selective, and cantransport a high number of drug molecules into the target cells. Inaddition, the intracellular release of the active agent is improved dueto reduction of the iron cation only within the endosomes. Undesiredrelease of the drug in the blood stream is thereby prevented or at leastsubstantially reduced.

The present invention therefore relates to, but is not limited to, theembodiments defined in the following items [1] to [43]:

-   -   [1] An antibody-drug conjugate comprising an antibody, a ferric        iron bound to the antibody, and at least one drug molecule bound        to the ferric iron.    -   [2] The antibody-drug conjugate of item [1], wherein the ferric        iron is complexed by the antibody and the drug molecule.    -   [3] The antibody-drug conjugate of item [1] or [2], wherein the        ferric iron is bound to the antibody via a first linker.    -   [4] The antibody-drug conjugate of item [3], wherein the first        linker comprises an iron complexing group capable of binding to        the ferric iron.    -   [5] The antibody-drug conjugate of item [3] or [4], wherein the        first linker is covalently bound to the antibody.    -   [6] The antibody-drug conjugate of any one of the preceding        items, wherein at least two drug molecules are bound to the        ferric iron.    -   [7] The antibody-drug conjugate of any one of the preceding        items, wherein the average number of drug molecules per antibody        in said conjugate is at least 5.    -   [8] The antibody-drug conjugate of any one of the preceding        items, wherein the average number of drug molecules per antibody        in said conjugate is at least 10.    -   [9] The antibody-drug conjugate of any one of the preceding        items, wherein the average number of drug molecules per antibody        in said conjugate is at least 15.    -   [10] The antibody-drug conjugate of any one of the preceding        items, wherein the drug molecule is not released from the        antibody-drug conjugate when the antibody-drug conjugate is in        human blood.    -   [11] The antibody-drug conjugate of any one of the preceding        items, wherein the drug molecule is released from the        antibody-drug conjugate when the antibody-drug conjugate is in        the endosomal compartment of a human cell.    -   [12] The antibody-drug conjugate of any one of the preceding        items, wherein the drug molecule is selected from the group        consisting of anti-cancer agents, anti-inflammatory agents, and        anti-infective agents.    -   [13] The antibody-drug conjugate of any one of the preceding        items, wherein the drug molecule is an anti-cancer agent.    -   [14] The antibody-drug conjugate of item [13], wherein the        anti-cancer agent is selected from the group consisting of        microtubule structure formation inhibitors, meiosis inhibitors,        RNA polymerase inhibitors, topoisomerase inhibitors, DNA        intercalating agents, DNA alkylating agents, and ribosome        inhibitors.    -   [15] The antibody-drug conjugate of any one of the preceding        items, wherein the antibody is capable of binding to an antigen        expressed on a tumor cell.    -   [16] The antibody-drug conjugate of any one of the preceding        items, wherein the drug molecule comprises an iron complexing        group which binds to the ferric iron.    -   [17] The antibody-drug conjugate of any one of items [1] to        [15], wherein the drug molecule is covalently linked to an iron        complexing group which binds to the ferric iron.    -   [18] The antibody-drug conjugate of item [16] or [17], wherein        said iron complexing group is selected from the group consisting        of pyridine derivatives, a hydroxamate group, a catecholate        group, a carboxylate group, and acids thereof.    -   [19] The antibody-drug conjugate of any one of the preceding        items, having the structure

Ab[-L1-Me (-L2-D)_(n)]_(m),

-   -    wherein        -   Ab is the antibody,        -   L1 is a first linker,        -   Me is the ferric iron,        -   L2 is a second linker or absent,        -   D is the drug molecule,        -   m is a number ranging from 1 to 10, and        -   n is a number ranging from 1 to 3.    -   [20] The antibody-drug conjugate of item [19], wherein m is a        number ranging from 2 to 6.    -   [21] The antibody-drug conjugate of item [19], wherein m is a        number ranging from 3 to 5.    -   [22] The antibody-drug conjugate of any one of items [19] to        [21], wherein n is 2 or 3.    -   [23] The antibody-drug conjugate of any one of the preceding        items, wherein the drug molecule is released from the ferric        iron if the ferric iron is reduced.    -   [24] The antibody-drug conjugate of any one of the preceding        items, wherein the drug molecule is released from the ferric        iron at a pH of less than 5.    -   [25] The antibody-drug conjugate of any one of the preceding        items, which is stable at pH 8.    -   [26] The antibody-drug conjugate of any one of the preceding        items, which is stable at pH 7.    -   [27] The antibody-drug conjugate of any one of the preceding        items, which is stable at pH 6.    -   [28] The antibody-drug conjugate of any one of the preceding        items, which is stable at pH 5.    -   [29] A pharmaceutical composition comprising the antibody-drug        conjugate of any one of the preceding items.    -   [30] The pharmaceutical composition of item [29], further        comprising a pharmaceutically acceptable excipient.    -   [31] The antibody-drug conjugate of any one of items [1] to [28]        for use as a therapeutic agent.    -   [32] The antibody-drug conjugate of any one of items [1] to [28]        for use in the treatment of a disorder.    -   [33] The antibody-drug conjugate of any one of items [1] to [28]        for use in the treatment of cancer.    -   [34] The antibody-drug conjugate for use according to item [33],        wherein said treatment comprises administering to a patient the        antibody-drug conjugate of any one of items [1] to [28] and an        anti-cancer agent different from said antibody-drug conjugate.    -   [35] Use of ferric iron for preventing or reducing the release        of a drug molecule from an antibody-drug conjugate in blood,        wherein the ferric iron is complexed by the antibody and the        drug molecule to form the antibody-drug conjugate.    -   [36] The use of item [35], wherein the drug molecule is released        from the antibody-drug conjugate in the endosomal compartment of        a cell.    -   [37] The use of item [35] or [36], wherein the antibody-drug        conjugate is the antibody-drug conjugate as defined in any one        of items [1] to [28].    -   [38] Use of the antibody-drug conjugate of any one of items [1]        to [28] for preventing or reducing the release of a drug        molecule from an antibody-drug conjugate in blood.    -   [39] A method of preventing or reducing the release of a drug        molecule from an antibody-drug conjugate in blood, comprising        complexing ferric iron with the antibody and the drug molecule        to form the antibody-drug conjugate.    -   [40] The method of item [39], wherein the antibody-drug        conjugate is the antibody-drug conjugate as defined in any one        of items [1] to [28].    -   [41] Use of ferric iron for linking a drug molecule to an        antibody.    -   [42] The use of item [41], wherein an antibody-drug conjugate is        obtained by said linking.    -   [43] The use of item [41] or [42], wherein said antibody-drug        conjugate is the antibody-drug conjugate as defined in any one        of items [1] to [28].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A New Concept for ADCs: Fe-Complex as a pH/Fe-reductase labilelinker. The cytotoxic compound is conjugated to ligands that complexiron. The complex is then conjugated to an antibody that has its naturalreceptor on the cancer cell. Upon antibody-antigen binding the entireADC is taken up by endocytosis and the complex disintegrates inside theendosome due to low pH and/or Fe-reductase activity, releasing the drug.Further action of, for example peptidases, may be required to releasethe active entity.

FIGS. 2A and 2B. The iron may be complexed by the cytotoxic drug itself.In this example, histone deacetylase inhibitors (e.g. SAHA (Vorinostat))are used to complex iron. This is an example for traceless drug releaseof iron-based ADCs. The hydroxamate functionality enables ironcomplexation through the drug itself without any modification of themolecule.

FIGS. 3A and 3B. The very potent drug Doxorubicin may also be used tocomplex iron after some modification of the amine function. Further modeof action is as described in FIG. 1.

FIGS. 4A and 4B. The upper graph (FIG. 4A) shows analytical LC-runs ofcomplex A at pH 5.0, at 37° C., over 16 h. The lower graph (FIG. 4B)shows analytical LC-runs of complex A at pH 8.0, at 37° C., over 16 h(see example 1).

FIG. 5. Overlay of analytical HPLC chromatograms of ligands used incomplexes A and B, their respective iron-complexes, and an additionalsample of preformed complex A with free ligand B, showing nointermediate complexes (example 2).

FIG. 6. Overlay of analytical HPLC chromatograms of free ligand (ST116),its respective complex and the hydrogen-reduced complex yielding therespective free ligand (example 3).

FIG. 7. Conjugation of an iron-complexing moiety to the AB, yieldingproduct AB-1, followed by iron complexation together with two auxiliaryligands (AB-2) (example 4).

FIGS. 8A and 8B. Upper graph (FIG. 8A): Fluorescence measurement ofconjugated/complexed AB-2 and the non-conjugated AB. Lower graph (FIG.8B): Difference in fluorescence of the complexed and non-complexedconjugates (example 4).

FIGS. 9A-9D. Fluorescence spectra of AB-2 (FIG. 9A) and its reducedderivative AB-1 (FIG. 9C), whereby the reduced complex gives ˜20% lessfluorescence (FIG. 9D—example 4). FIG. 9B depicts the non-conjugatedantibody (AB) in isolation.

FIGS. 10A-10C. Fluorescence spectra of a non-reduced ST119 conjugate(FIG. 10A) and its reduced derivative (FIG. 10B) are presented in FIG.10C. Fluorescence of N-Hydroxamate Ornithine coupled to Fluoresceinamine (ST102) is quenched by Fe³⁺ but regains fluorescence uponreduction of Fe³⁺ to Fe²⁺ (example 5).

DETAILED DESCRIPTION

In a first aspect, the invention relates to an antibody-drug conjugatecomprising an antibody, at least one ferric iron bound to the antibody,and at least one drug molecule bound to the ferric iron.

Antibody

As used herein, the term “antibody” refers to an immunoglobulin (Ig),which is defined as a protein belonging to the class IgG, IgM, IgE, IgA,or IgD (or any subclass thereof), or a functional fragment thereof. Inthe context of the present invention, a “functional fragment” of animmunoglobulin is defined as antigen-binding fragment or otherderivative of a parental immunoglobulin that essentially maintains theantigen binding activity of such parental immunoglobulin. Functionalfragments are antibodies in the sense of the present invention even iftheir affinity to the antigen is lower than that of the parentalimmunoglobulin. “Functional fragments” in accordance with the inventioninclude F(ab′)₂ fragments, Fab fragments, scFv, dsFv, diabodies,triabodies, tetrabodies and Fc fusion proteins. The F(ab′)₂ or Fab maybe engineered to minimize or completely remove the intermoleculardisulphide interactions that occur between the CH1 and CL domains. Theantibodies of the present invention may be part of bi- ormultifunctional constructs. The antibodies of the present inventioninclude, but are not limited to, monoclonal antibodies, humanantibodies, humanized antibodies, chimeric antibodies, andanti-idiotypic antibodies.

Preferably, the antibody of the present invention is a monoclonalantibody.

The antibody of the ADC of the present invention preferably specificallybinds to an antigen expressed on the surface of a cancer cell.

Drug Molecule

The term “drug molecule” or “payload” as used herein refers to atherapeutic or diagnostic agent. Preferred drug molecules includeanti-cancer agents, anti-inflammatory agents, and anti-infective (e.g.,anti-fungal, antibacterial, anti-parasitic, anti-viral) agents.

Preferably, the drug molecule of the present invention is an anti-canceragent. Suitable anti-cancer agents include, but are not limited to,alkylating agents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors,photosensitizers, and kinase inhibitors.

Also included in the definition of “anti-cancer agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptormodulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands; (iii)anti-androgens; (iv) protein kinase inhibitors; (v) lipid kinaseinhibitors; (vi) antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in aberrantcell proliferation; (vii) ribozymes such as VEGF expression inhibitorsand HER2 expression inhibitors; (viii) vaccines such as gene therapyvaccines; topoisomerase 1 inhibitors; (ix) anti-angiogenic agents; andpharmaceutically acceptable salts, acids, solvates and derivatives ofany of the above.

The most preferred anti-cancer agents in the present invention are thosethat can be removed nearly traceless upon disintegration of theiron-ligand complex. Meaning that compounds with high binding affinitiesto their target require no or minor modification in order to complexwith iron. Most notable in the presented example are compounds such asthe HDAc inhibitor ST-3595 (see FIGS. 2A and 2B). However, alsodifferent compounds may also be highly suitable to complex with ferriciron while being able to disintegrate upon iron reduction/ligandprotonation.

Ferric Iron

The term “ferric iron” as used herein refers to an iron cation with anoxidation number of +3, also designated iron(III), Fe(III) or Fe³⁺.

Generally, pharmaceutically acceptable transition metal cations that arecapable of forming a complex with other compounds include, for example,cations of platinum, ruthenium, iridium, scandium, titanium, vanadium,chromium, manganese, iron, cobalt, nickel, copper and zinc. Thetransition metal cation in the ADC of the invention, however, is ferriciron.

Conjugate

Typically, the ferric iron is indirectly bound to the antibody via afirst linker.

The term “linker”, as used herein, refers to a chemical moiety whichattaches a molecule or an atom to a chemical compound, e.g. to anantibody or to a drug molecule. Typically, the linker comprises a chainof atoms linked by chemical bonds.

The first linker attaches the ferric iron to the antibody.

One end of the first linker is preferably covalently attached to theantibody. The antibody-reactive end of the first linker is typically asite that is capable of conjugation to the antibody through a cysteinethiol or lysine amine group on the antibody, and so is typically athiol-reactive group such as a double bond (as in maleimide) or aleaving group such as a chloro, bromo, or iodo, or an R-sulfanyl group,or an amine-reactive group such as a carboxyl group. When the term“linker” is used in describing the linker in conjugated form, onereactive end will be absent (such as the leaving group of athiol-reactive group) or incomplete (such as there being only thecarbonyl of the carboxylic acid) because of the formation of the bondsbetween the linker and the antibody. Various linker types are describedin McCombs et al. (2015) The AAPS Journal, Vol. 17, No. 2, pages339-351, and in Lu et al. (2016) Int. J. Mol. Sci. 17, 561(doi:10.3390/ijms17040561) the content of which is incorporated hereinby reference.

Linkers can comprise a variety of chemical groups which include, but arenot limited to, optionally substituted divalent radicals such asalkylene, arylene, heteroarylene; moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino); anddiacid ester and amides including succinate, succinamide, diglycolate,malonate, and caproamide; wherein each R is independently H,C₁-C₁₈alkyl, C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, a protecting group or aprodrug moiety, and n is an integer from 1 to 10.

In a specific embodiment, the linker may be peptidic, comprising one ormore amino acid units. Examples of amino acid linkers include adipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Amino acidlinker components may or may not be designed and optimized in theirselectivity for enzymatic cleavage by a particular enzyme.

The second end of the first linker preferably comprises or consists ofan iron complexing group which is capable of binding to the ferric ironof the ADC. The iron complexing group is preferably a chelating group.More preferably, the iron complexing group is a chelating group of asiderophore. In a preferred embodiment, the iron complexing group is acatecholate group, a carboxylate group, or a hydroxamate group, as shownin the following formulae (I) to (III), respectively, wherein R is therest of the linker including the first end.

As used herein, the terms catecholate group, carboxylate group, andhydroxamate group include the respective acid forms and deprotonatedforms thereof.

A preferred first linker of the ADC of the invention is a compound offormula (I), wherein R is —(CH₂)_(p)—CH(NHR²)—(C═O) . . . , p is aninteger from 0 to 6, R² is a formyl group, an acetyl group, a peptidylgroup, or a C₁-C₂₀ hydrocarbon group, and wherein the dotted lineindicates the binding site to the antibody. Preferably, p is an integerfrom 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular first linker of the ADC of the invention is a compound offormula (I), wherein R is —(CH₂)_(p)—CH(NH₂)—(C═O) . . . , p is aninteger from 0 to 6, and the dotted line indicates the binding site tothe antibody. Preferably, p is an integer from 2 to 5, more preferably pis 3 or 4, most preferably p is 3.

Another preferred first linker of the ADC of the invention is a compoundof formula (I), wherein R is —C═O—NH—(CH₂)_(p)—CH(NHR²)—(C═O) . . . , pis an integer from 0 to 6, R² is a formyl group, an acetyl group, apeptidyl group, or a C₁-C₂₀ hydrocarbon group, and the dotted lineindicates the binding site to the antibody. Preferably, p is an integerfrom 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another particular first linker of the ADC of the invention is acompound of formula (I), wherein R is —C═O—NH—(CH₂)_(p)—CH(NH₂)—(C═O) .. . , p is an integer from 0 to 6, and the dotted line indicates thebinding site to the antibody. Preferably, p is an integer from 2 to 5,more preferably p is 3 or 4, most preferably p is 3.

A preferred first linker of the ADC of the invention is a compound offormula (II), wherein R is —(CH₂)_(p)—CH(NHR²)—(C═O) . . . , p is aninteger from 0 to 6, R² is a formyl group, an acetyl group, a peptidylgroup, or a C₁-C₂₀ hydrocarbon group, and wherein the dotted lineindicates the binding site to the antibody. Preferably, p is an integerfrom 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular first linker of the ADC of the invention is a compound offormula (II), wherein R is —(CH₂)_(p)—CH(NH₂)—(C═O) . . . , p is aninteger from 0 to 6, and the dotted line indicates the binding site tothe antibody. Preferably, p is an integer from 2 to 5, more preferably pis 3 or 4, most preferably p is 3.

Another preferred first linker of the ADC of the invention is a compoundof formula (II), wherein R is —C═O—NH—(CH₂)_(p)—CH(NHR²)—(C═O) . . . , pis an integer from 0 to 6, R² is a formyl group, an acetyl group, apeptidyl group, or a C₁-C₂₀ hydrocarbon group, and the dotted lineindicates the binding site to the antibody. Preferably, p is an integerfrom 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another particular first linker of the ADC of the invention is acompound of formula (II), wherein R is —C═O—NH—(CH₂)_(p)—CH(NH₂)—(C═O) .. . , p is an integer from 0 to 6, and the dotted line indicates thebinding site to the antibody. Preferably, p is an integer from 2 to 5,more preferably p is 3 or 4, most preferably p is 3.

A preferred first linker of the ADC of the invention is a compound offormula (III), wherein R is —(CH₂)_(p)—CH(NHR²)—(C═O) . . . , p is aninteger from 0 to 6, R² is a formyl group, an acetyl group, a peptidylgroup, or a C₁-C₂₀ hydrocarbon group, and wherein the dotted lineindicates the binding site to the antibody. Preferably, p is an integerfrom 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular first linker of the ADC of the invention is a compound offormula (III), wherein R is —(CH₂)_(p)—CH(NH₂)—(C═O) . . . , p is aninteger from 0 to 6, and the dotted line indicates the binding site tothe antibody. Preferably, p is an integer from 2 to 5, more preferably pis 3 or 4, most preferably p is 3.

Another preferred first linker of the ADC of the invention is a compoundof formula (III), wherein R is —C═O—NH—(CH₂)_(p)—CH(NHR²)—(C═O) . . . ,p is an integer from 0 to 6, R² is a formyl group, an acetyl group, apeptidyl group, or a C₁-C₂₀ hydrocarbon group, and the dotted lineindicates the binding site to the antibody. Preferably, p is an integerfrom 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another particular first linker of the ADC of the invention is acompound of formula (III), wherein R is —C═O—NH—(CH₂)_(p)—CH(NH₂)—(C═O). . . , p is an integer from 0 to 6, and the dotted line indicates thebinding site to the antibody. Preferably, p is an integer from 2 to 5,more preferably p is 3 or 4, most preferably p is 3.

In other preferred embodiments of the invention the iron complexinggroup is a pyridine derivative which is capable of complexing a ferriciron as defined hereinabove. Suitable pyridine derivatives are describedin, e.g. “The Chemistry of Heterocyclic Compounds, Pyridine MetalComplexes”, ed. Desmond J. Brown, John Wiley & Sons, 2009 (ISBN-13:9780470239728), the disclosure of which is incorporated herein in itsentirety. The embodiments described above in connection with Formula(I), (II) or (III) are applicable to other chelating agent described inthis reference mutatis mutandis.

Further suitable iron complexing groups are described in “ChelatingAgents and Metal Chelates”, ed. F Dwyer, 2012 (ISBN-13: 9780323146418),the disclosure of which is incorporated herein in its entirety. Theembodiments described above in connection with Formula (I), (II) or(III) are applicable to other chelating agent described in thisreference mutatis mutandis.

The average number of first linkers per antibody (i.e. the molar “firstlinker-to-antibody ratio”) in the ADC of the invention is typically atleast 1, preferably at least 2, more preferably at least 5, morepreferably at least 8, more preferably at least 10, most preferably >10.

The molar “first linker-to-antibody ratio” in the ADC of the inventionis typically 1 to 50, preferably 2 to 40, more preferably 5 to 35, morepreferably 8 to 30, most preferably 10 to 20, e.g. 11 to 20, 12 to 20,13 to 20, 14 to 20, or 15 to 20. In other embodiments the molar “firstlinker-to-antibody ratio” is 10 to 19, or 11 to 18, or 12 to 17, or 13to 16, e.g.13, 14, 15 or 16.

The molar “first linker-to-antibody ratio” in the ADC of the inventionis preferably >10.

The ADC of the present invention can comprise one or more ferric ironsper antibody molecule. The number of ferric irons per antibody molecule(i.e. the molar “ferric iron-to-antibody ratio”) is preferably equal tothe number of first linkers that were conjugated to the antibody, incase the first linker is capable of complexing only one ferric iron.Depending on the chemical design, the first linker may be capable ofcomplexing more than one ferric iron, e.g. 2 or 3 ions (see Formulabelow).

The average number of drug molecules per antibody (i.e. the molar“drug-to-antibody ratio”) in the ADC of the invention usually is twicethe number of first linkers that are conjugated to the antibody. Hence,typically from 2 to 30 or from 4 to 20. In the example in the formulaabove, one first linker serves to complex 3 ferric ions, with 2*3=6concomitant drug molecules. In the ADC of the invention the drugmolecule is directly or indirectly bound to the ferric iron. The bindingis preferably effected via iron complexing groups as defined above forthe first linker.

If the drug molecule is directly bound to the ferric iron, the drugmolecule itself is capable of binding to the ferric iron. According tothis embodiment the drug molecule preferably comprises an ironcomplexing group. The iron complexing groups may or may not be the sameas defined above for the first linker. Examples of drug molecules thatare capable of binding to the ferric iron include the following HDACinhibitors:

As can be seen, the drug molecules comprise a hydroxamate group which iscapable of binding to a ferric iron. This is also illustrated in FIGS.2A and 2B. FIG. 3A also shows an embodiment where the drug molecule(doxorubicin) is modified to allow binding to the ferric iron. As thereleased modified drug molecule is active, this is still considered“direct binding” of the drug molecule to the ferric iron.

If the drug molecule is indirectly bound to the ferric iron the drugmolecule is attached to a second linker which comprises a chemical groupthat is capable of binding to the ferric iron. The chemical group thatis capable of binding to the ferric iron typically is a metal complexinggroup that is capable of binding to the ferric iron. The preferred ironcomplexing groups described above in connection with the first linkerare also preferred iron complexing groups of the second linker. That is,one end of the second linker preferably comprises or consists of ahydroxamate group, a catecholate group or a carboxylate group. The otherend of the second linker is preferably covalently attached to the drugmolecule. This embodiment is illustrated in FIG. 1, wherein the drugmolecule is designated “payload”. FIG. 3B also shows an embodiment wheredoxorubicin is attached to a linker which comprises an iron complexinggroup so as to allow binding to the ferric iron.

A preferred second linker of the ADC of the invention is a compound offormula (I), wherein R is —(CH₂)_(p)—CH(NHR²)—(C═O) . . . , p is aninteger from 0 to 6, R² is a formyl group, an acetyl group, a peptidylgroup, or a C₁-C₂₀ hydrocarbon group, and wherein the dotted lineindicates the binding site to the drug molecule. Preferably, p is aninteger from 2 to 5, more preferably p is 3 or 4, most preferably p is3.

A particular second linker of the ADC of the invention is a compound offormula (I), wherein R is —(CH₂)_(p)—CH(NH₂)—(C═O) . . . , p is aninteger from 0 to 6, and the dotted line indicates the binding site tothe drug molecule. Preferably, p is an integer from 2 to 5, morepreferably p is 3 or 4, most preferably p is 3.

Another preferred second linker of the ADC of the invention is acompound of formula (I), wherein R is —C═O—NH—(CH₂)_(p)—CH(NHR²)—(C═O) .. . , p is an integer from 0 to 6, R² is a formyl group, an acetylgroup, a peptidyl group, or a C₁-C₂₀ hydrocarbon group, and the dottedline indicates the binding site to the drug molecule. Preferably, p isan integer from 2 to 5, more preferably p is 3 or 4, most preferably pis 3.

Another particular second linker of the ADC of the invention is acompound of formula (I), wherein R is —C═O—NH—(CH₂)_(p)—CH(NH₂)—(C═O) .. . , p is an integer from 0 to 6, and the dotted line indicates thebinding site to the drug molecule. Preferably, p is an integer from 2 to5, more preferably p is 3 or 4, most preferably p is 3.

A preferred second linker of the ADC of the invention is a compound offormula (II), wherein R is —(CH₂)_(p)—CH(NHR²)—(C═O) . . . , p is aninteger from 0 to 6, R² is a formyl group, an acetyl group, a peptidylgroup, or a C₁-C₂₀ hydrocarbon group, and wherein the dotted lineindicates the binding site to the drug molecule. Preferably, p is aninteger from 2 to 5, more preferably p is 3 or 4, most preferably p is3.

A particular second linker of the ADC of the invention is a compound offormula (II), wherein R is —(CH₂)_(p)—CH(NH₂)—(C═O) . . . , p is aninteger from 0 to 6, and the dotted line indicates the binding site tothe drug molecule. Preferably, p is an integer from 2 to 5, morepreferably p is 3 or 4, most preferably p is 3.

Another preferred second linker of the ADC of the invention is acompound of formula (II), wherein R is —C═O—NH—(CH₂)_(p)—CH(NHR²)—(C═O). . . , p is an integer from 0 to 6, R² is a formyl group, an acetylgroup, a peptidyl group, or a C₁-C₂₀ hydrocarbon group, and the dottedline indicates the binding site to the drug molecule. Preferably, p isan integer from 2 to 5, more preferably p is 3 or 4, most preferably pis 3.

Another particular second linker of the ADC of the invention is acompound of formula (II), wherein R is —C═O—NH—(CH₂)_(p)—CH(NH₂)—(C═O) .. . , p is an integer from 0 to 6, and the dotted line indicates thebinding site to the drug molecule. Preferably, p is an integer from 2 to5, more preferably p is 3 or 4, most preferably p is 3.

A preferred second linker of the ADC of the invention is a compound offormula (III), wherein R is —(CH₂)_(p)—CH(NHR²)—(C═O) . . . , p is aninteger from 0 to 6, R² is a formyl group, an acetyl group, a peptidylgroup, or a C₁-C₂₀ hydrocarbon group, and wherein the dotted lineindicates the binding site to the drug molecule. Preferably, p is aninteger from 2 to 5, more preferably p is 3 or 4, most preferably p is3.

A particular second linker of the ADC of the invention is a compound offormula (III), wherein R is —(CH₂)_(p)—CH(NH₂)—(C═O) . . . , p is aninteger from 0 to 6, and the dotted line indicates the binding site tothe drug molecule. Preferably, p is an integer from 2 to 5, morepreferably p is 3 or 4, most preferably p is 3.

Another preferred second linker of the ADC of the invention is acompound of formula (III), wherein R is —C═O—NH—(CH₂)_(p)—CH(NHR²)—(C═O). . . , p is an integer from 0 to 6, R² is a formyl group, an acetylgroup, a peptidyl group, or a C₁-C₂₀ hydrocarbon group, and the dottedline indicates the binding site to the drug molecule. Preferably, p isan integer from 2 to 5, more preferably p is 3 or 4, most preferably pis 3.

Another particular second linker of the ADC of the invention is acompound of formula (III), wherein R is —C═O—NH—(CH₂)_(p)—CH(NH₂)—(C═O). . . , p is an integer from 0 to 6, and the dotted line indicates thebinding site to the drug molecule. Preferably, p is an integer from 2 to5, more preferably p is 3 or 4, most preferably p is 3.

In one embodiment, the second linker of the ADC of the invention is anon-cleavable linker. Examples of non-cleavable linkers aremaleimide-alkane linkers (e.g. maleimido-caproyl linkers) andmaleimide-cyclohexane linkers.

In another embodiment, the second linker of the ADC of the invention isa cleavable linker. Cleavable linkers include chemically labile linkersand enzyme cleavable linkers. The chemically labile linker can be anacid-cleavable linker or a reducible linker. The acid-cleavable linkermay comprise a hydrazone group. The reducible linker may comprise adisulfide group. The enzyme cleavable linker typically comprises achemical group which can be cleaved or degraded by one or more lysosomalenzymes. Suitable groups include a valine-citrulline dipeptide group, aphenylalanine-lysine dipeptide group, and a β-glucuronide group (whichcan be cleaved by β-glucuronidase).

In a specific embodiment of the invention, the first linker is anon-cleavable linker, and the second linker is a cleavable linker. Inanother specific embodiment of the invention, the first linker is anon-cleavable linker, and the second linker is a non-cleavable linker.In another specific embodiment of the invention, the first linker is acleavable linker, and the second linker is a cleavable linker. Inanother specific embodiment of the invention, the first linker is acleavable linker, and the second linker is a non-cleavable linker.

The drug molecule is released from the ADC upon internalization of theADC by the target cell, preferably a cancer cell. Without wishing to bebound by theory, it is believed that the release is enhanced by a lowpH, and by reduction of the ferric iron, e.g. by the enzyme ferricreductase. Due to the lower pH inside endosomes and increased presenceof ferric reductase, an Fe³⁺-based ADC complex should decompose insidethe endosome, enhancing release or diffusion of the smaller ligands tothe intracellular space.

In one embodiment the drug molecule is released from the ADC of theinvention when the ADC is in the endosomal compartment of a cell,preferably a mammalian cell, most preferably a human cell.

Preferably, at least 25%, more preferably at least 50%, more preferablyat least 75%, most preferably at least 95% of the drug molecules arereleased from the ADC upon reduction of the ferric iron to ferrous iron,as determined in an assay as described in Example 3.

In another embodiment, the ADC of the invention is stable at a pH of 8,as determined in an assay as described in Example 1. In anotherembodiment, the ADC of the invention is stable at a pH of 7, asdetermined in an assay as described in Example 1. In another embodiment,the ADC of the invention is stable at a pH of 6, as determined in anassay as described in Example 1. In another embodiment, the ADC of theinvention is stable at a pH of 5, as determined in an assay as describedin Example 1. “Stable” in the sense of this assay means that incubationof the complex under the conditions of example 1 (pH 5 and pH 8) doesnot lead to release of free ligand which would be seen as a second peakin a graph according to FIGS. 4A and 4B.

Preferably, the drug molecule is not released from the ADC of theinvention when the antibody is in blood, preferably in human blood. Inother embodiments the drug molecule is not released from the ADC of theinvention when the antibody is in blood plasma, preferably blood plasmafrom a vertebrate, more preferably human blood plasma.

In yet another embodiment, the ADC of the invention shows substantiallyno ligand exchange, as determined in an assay as described in Example 2.

Another aspect of the invention is an ADC having the followingstructure:

Ab[-L1-Me (-L2-D)_(n)]_(m),

-   -   wherein    -   Ab is an antibody,    -   L1 is a first linker,    -   Me is ferric iron,    -   L2 is a second linker or absent,    -   D is a drug molecule,    -   m is from 1 to 10, and    -   n is from 1 to 3.

The preferred embodiments of antibody, first and second linker, ferriciron and drug molecule described above apply to this aspect of theinvention mutatis mutandis. The parameter m is preferably from 2 to 6,or 3 to 5, e.g. 3, 4, or 5. The parameter n is preferably 2 or 3, mostpreferably 2. In a special embodiment m and/or n are/is an integer.

In a preferred embodiment n is 2 and m is 2.

In another preferred embodiment n is 2 and m is 3.

In another preferred embodiment n is 2 and m is 4.

In another preferred embodiment n is 2 and m is 5.

In another preferred embodiment n is 2 and m is 6.

In another preferred embodiment n is 2 and m is 7.

In another preferred embodiment n is 2 and m is 8.

In another preferred embodiment n is 2 and m is 9.

In another preferred embodiment n is 2 and m is 10.

In another preferred embodiment n is 3 and m is 2.

In another preferred embodiment n is 3 and m is 4.

In another preferred embodiment n is 3 and m is 5.

In another preferred embodiment n is 3 and m is 6.

In another preferred embodiment n is 3 and m is 7.

In another preferred embodiment n is 3 and m is 8.

In another preferred embodiment n is 3 and m is 9.

In another preferred embodiment n is 3 and m is 10.

Preparation of the ADC

Another aspect of the invention is a method for preparing the ADC of thepresent invention. The linkers can be synthesized by methods comprisingsteps that are known per se.

The ADC of the invention may be prepared as described in the following:

In the present invention, as described in example 5, an iron-complexingmoiety is covalently attached to an antigen-recognizing structure (e.g.antibody) of interest. The most straightforward way is via succinimideactivated conjugation as employed in example 5. However, a multitude ofconjugation strategies are described in literature (e.g. maleinimide andclick-chemistry methods). Following the conjugation of the first linker,the coupled product is purified and subsequently incubated respectivelywith a) two equivalents of a payload (this being an iron-complexingcompound or any compound that is linked to an iron-complexing moiety)and b) one equivalent of an iron salt (in the case of example 5 this isFeCl₃). The resulting product is then purified again using a sizeexclusion column.

Treatment

In one embodiment, the invention provides a method of treating orpreventing a disease comprising administering the ADCs of the inventionto a patient, preferably a human patient. In certain embodiments, thedisease to be treated or prevented is a cancer.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric or stomach cancer including gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectalcancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, aswell as head and neck cancer.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight.

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the ADC. Other dosageregimens may be useful. The progress of this therapy is easily monitoredby conventional techniques and assays.

Combination Therapy

An antibody-drug conjugate (ADC) may be combined in a pharmaceuticalcombination formulation, or dosing regimen as combination therapy, witha second compound having anti-cancer properties. The second compound ofthe pharmaceutical combination formulation or dosing regimen preferablyhas complementary activities to the ADC of the combination such thatthey do not adversely affect each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent,cytokine, growth inhibitory agent, anti-hormonal agent, aromataseinhibitor, protein kinase inhibitor, lipid kinase inhibitor,anti-androgen, antisense oligonucleotide, ribozyme, gene therapyvaccine, anti-angiogenic agent and/or cardioprotectant. Such moleculesare suitably present in combination in amounts that are effective forthe purpose intended. A pharmaceutical composition containing an ADC mayalso have a therapeutically effective amount of a chemotherapeutic agentsuch as a tubulin-forming inhibitor, a topoisomerase inhibitor, or a DNAbinder.

Other therapeutic regimens may be combined with the administration of ananticancer agent identified in accordance with this invention. Thecombination therapy may be administered as a simultaneous or sequentialregimen. When administered sequentially, the combination may beadministered in two or more administrations. The combined administrationincludes co-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein there is a time period while both (or all) active agentssimultaneously exert their biological activities.

The combination therapy may provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Also falling within the scope of this invention are the in vivometabolic products of the ADC compounds described herein, to the extentsuch products are novel and unobvious over the prior art. Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification, enzymatic cleavage, and the like, of theadministered compound. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolites include the products of in vivo cleavage of the ADC wherecleavage of any bond occurs that links the drug moiety to the antibody.Metabolic cleavage may thus result in the naked antibody, or an antibodyfragment. The antibody metabolite may be linked to a part, or all, ofthe linker. Metabolic cleavage may also result in the production a drugmoiety or part thereof. The drug moiety metabolite may be linked to apart, or all, of the linker.

Administration of Antibody-Drug Conjugate Pharmaceutical Formulations

Therapeutic ADCs may be administered by any route appropriate to thecondition to be treated. The ADC will typically be administeredparenterally, i.e. infusion, subcutaneous, intramuscular, intravenous,intradermal, intrathecal, bolus, intratumor injection or epidural (Shireet al (2004) J. Pharm. Sciences 93(6):1390-1402). Pharmaceuticalformulations of therapeutic antibody-drug conjugates (ADC) are typicallyprepared for parenteral administration with a pharmaceuticallyacceptable parenteral vehicle and in a unit dosage injectable form. Anantibody-drug conjugate (ADC) having the desired degree of purity isoptionally mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers, in the form of a lyophilized formulation oran aqueous solution (Remington's Pharmaceutical Sciences (1980) 16thedition, Osol, A. Ed.).

Acceptable parenteral vehicles, diluents, carriers, excipients, andstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). For example, lyophilized anti-ErbB2 antibodyformulations are described in WO 97/0480.

Pharmaceutical formulations of a therapeutic antibody-drug conjugate(ADC) may contain certain amounts of unreacted drug moiety (D),antibody-linker intermediate (Ab-L), and/or drug-linker intermediate(D-L), as a consequence of incomplete purification and separation ofexcess reagents, impurities, and by-products, in the process of makingthe ADC; or time/temperature hydrolysis or degradation upon storage ofthe bulk ADC or formulated ADC composition. For example, a formulationof the ADC may contain a detectable amount of free drug D.Alternatively, or in addition to, it may contain a detectable amount ofdrug-linker intermediate D-L. Alternatively, or in addition to, it maycontain a detectable amount of the antibody, Ab. An exemplaryformulation of may contain up to 10% molar equivalent of free drug.

The active pharmaceutical ingredients may also be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi permeable matrices of solidhydrophobic polymers containing the ADC, which matrices are in the formof shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile,which is readily accomplished by filtration through sterile filtrationmembranes.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Aqueous suspensions contain the active materials (ADC) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose or saccharin.

The pharmaceutical compositions of ADC may be in the form of a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, anaqueous solution intended for intravenous infusion may contain fromabout 3 to 500 μg of the active ingredient per milliliter of solution inorder that infusion of a suitable volume at a rate of about 30 mL/hr canoccur. Subcutaneous (bolus) administration may be effected with about1.5 ml or less of total volume and a concentration of about 100 mg ADCper ml. For ADC that require frequent and chronic administration, thesubcutaneous route may be employed, such as by pre-filled syringe orautoinjector device technology.

As a general proposition, the initial pharmaceutically effective amountof ADC administered per dose will be in the range of about 0.01-100mg/kg, namely about 0.1 to 20 mg/kg of patient body weight per day, withthe typical initial range of compound used being 0.3 to 15 mg/kg/day.For example, human patients may be initially dosed at about 1.5 mg ADCper kg patient body weight. The dose may be escalated to the maximallytolerated dose (MTD). The dosing schedule may be about every 3 weeks,but according to diagnosed condition or response, the schedule may bemore or less frequent. The dose may be further adjusted during thecourse of treatment to be at or below MTD which can be safelyadministered for multiple cycles, such as about 4 or more.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Although oral administration of protein therapeutics are generallydisfavored due to poor bioavailability due to limited absorption,hydrolysis or denaturation in the gut, formulations of ADC suitable fororal administration may be prepared as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the ADC.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Exemplary unit dosage formulations contain a dailydose or unit daily sub-dose, or an appropriate fraction thereof, of theactive ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

A further aspect of the present invention is the use of the ADCdescribed herein for preventing or reducing the release of a drugmolecule from an ADC in blood or plasma, preferably in human blood orplasma. Another aspect of the invention is the use of ferric iron forpreventing or reducing the release of a drug molecule from anantibody-drug conjugate in blood or plasma, preferably in human blood orplasma, wherein the ferric iron is complexed by the antibody and thedrug molecule to form the antibody-drug conjugate. Another aspect of theinvention is a method of preventing or reducing the release of a drugmolecule from an antibody-drug conjugate in blood, comprising complexingferric iron with the antibody and the drug molecule to form theantibody-drug conjugate. The preferred embodiments of the uses andmethods of the invention correspond to those of the ADC of the inventionas described hereinabove.

EXAMPLES Example 1

To assess the stability of the complexes that we intended to use for theabove described linkers, a number of experiments were conducted. To thisend, two model-ligands (GVFe35 and ST088) were synthesized (See Scheme1).

Using these two ligands, the following two model complexes were preparedby addition of FeCl₃ solution.

The formulae show the structures of iron complexes formed using GVFe35(free carbonic acid, Complex A) and ST088 (sec-butylamine coupled,Complex B).

Assessing the complex stability at different pHs over time, Complex Awas then incubated at pH 5.0 or pH 8.0 at 37° C. Eventual degradation ofthe complex was monitored by analytical HPLC at regular time-intervalsand after 16 h. The HPLC-chromatograms are depicted in FIGS. 4A and 4B.It follows, that after 16 h only modest degradation could be observed inboth cases, indicating that under these conditions the complexes arestable.

Example 2

To examine if ligand exchange occurs (i.e. ligand dissociation andrenewed complexation), an additional experiment was conducted, wherebyboth complexes A and B were mixed and incubated at 37° C. at pH 7.0 for16 h. In case of dissociation and subsequent complexation one wouldexpect intermediate complexes, whereby one or two ligands of complex Bwould complex iron together with one ligand stemming from complex A orvice versa.

Both complexes and the mixture were examined by analytical HPLC (SeeFIG. 5). Due to the more apolar sec-butyl amide function in ST088, thatwas used for complex B, this complex has a longer retention time thancomplex A. Therefore, in case of ligand exchange, peaks withintermediate retention time would start occurring between the twocomplex peaks. Since no intermediate peaks were observed, this suggeststhat no ligand exchange occurs for the examined complexes, indicatinghigh stability.

Example 3

To further corroborate this result and to examine the influence of theirons oxidation state, further experiments were conducted. Free ligandwas mixed with FeCl₃, giving complex A that was subsequently reduced byhydrogenation:

Ligand, complex A, and reduced material were analyzed by analytical LC(See FIG. 6). It is shown that complex A has a longer retention timethan the free ligand. However, after reduction, only a peak with thesame retention time as the native ligand shows. From this, it must beconcluded that the reduced complex is destabilized, leading todecomplexation which in turn results in un-complexed ferrous iron andthe free ligands. A complementary experiment showed respective resultsfor the oxidation of Fe(II) in the presence of free ligand and air.

Example 4

For further studies of antibodies to which iron complexes areconjugated, a fluorescently Rhodamine 101) labeled derivative of alysine based hydroxamate was synthesized (see Scheme 2).

This ligand was next conjugated to Trastuzumab for further complexationtogether with the non-OSu activated ligand, which is also coupled toRhodamine 101 (see FIG. 7).

The AB-conjugated complex was purified by centrifugation in Vivaspintubes (cut-off <30 kDa), by washing the conjugated complex twice withcitrate buffer (5 mL). This was followed by diluting the product to 0.5mL and fluorescence measurement. From FIGS. 8A and 8B, it follows, thatAB-2 yields more fluorescence than the non-conjugated AB, and AB-1 lessthan AB-2. A significant difference in fluorescence for both compoundsis observed, showing that complexation with iron and the two auxiliaryligands was successful (see FIG. 8B).

To examine if the complex can disintegrate by destabilization due toreduction of the ferric form of the complex into the ferrous complex,AB-2 (FIG. 9A) was reduced to obtain AB-1 (FIG. 9C). To this end, AB-2was reduced under hydrogen atmosphere for 1 h at RT and purified byVivaspin centrifugation and washed twice with 5 mL citrate buffer. Thiswas followed by fluorescence measurement (see FIG. 9D), from which itcan be observed that the control, that underwent the same procedure(except hydrogenation), shows again ˜20% more fluorescence than thereduced product. This shows that AB-2 was reduced to give AB-1.

Example 5

To assess whether an antibody conjugate, containing a payload that isbound via ferric iron to the antybody, would a) actually be endocytosedinto cells expressing the appropriate antigen, and b) retains sufficientstability in proximity of cell membranes in physiological medium, atrastuzumab conjugate as depicted in Scheme 3 was prepared. As a firstlinker, a lysine-based hydroxamate that was also conjugated to BODIPYwas used. The hydroxamate moiety of this structure is able to complexferric iron with two auxiliary ligands. As auxiliary ligands, againlysine-based hydroxamates were used that in this case were conjugated toRhodamine 101. Hence, a trastuzumab conjugate was created that containeda covalently linked BODIPY tag, capable of complexing ferric iron, aswell as two Rhodamine containing ligands that are non-covalentlyconjugated to the antibody via the ferric iron complex. See FIGS. 10Aand 10B.

SK-BR3-Cells were grown overnight on coverslips withPolyornithincoating. Subsequently these cells were incubated withGVFe66b up to 30 minutes or 6 h and washed twice with PBS buffer andfixated (PFA).

Fluorescence microscopy after 30 minutes and six hours shows both theBODIPY and rhodamine signal on the cell surface after one hour and inendosomal compartments after six hours. From this observation it must beconcluded that the conjugate retains certain stability up tointernalization. See FIG. 10C.

Scheme 3. Preparation of a Trastuzumab conjugated ADC, using a ferriciron linker concept. Compound GVFe49 was prepared by standard literaturebased synthesis. Subsequently, GVFe49 was conjugated to commerciallyavailable BODIPY-NHS, yielding compound GVFe60, which was nextdeprotected, yielding GVFe61, and OSu activated, yielding GVFe62. Thiscompound was next used to conjugate to the antibody. Subsequently, thisconjugate was incubated with FeCl₃ and compound GVFe54 (which was inturn synthesized via the same method as described for compound GVFe49).

1. An antibody-drug conjugate comprising an antibody, at least oneferric iron bound to the antibody, and at least one drug molecule boundto the ferric iron.
 2. The antibody-drug conjugate of claim 1, whereinthe drug molecule is not released from the antibody-drug conjugate whenthe antibody-drug conjugate is in blood.
 3. The antibody-drug conjugateof claim 1, wherein the drug molecule is released from the antibody-drugconjugate when the antibody-drug conjugate is in the endosomalcompartment of a cell.
 4. The antibody-drug conjugate of claim 1,wherein at least two drug molecules are bound to one ferric iron.
 5. Theantibody-drug conjugate of claim 1, wherein the average number of drugmolecules per antibody in said conjugate is at least
 10. 6. Theantibody-drug conjugate of claim 1, wherein the drug molecule is ananti-cancer agent.
 7. The antibody-drug conjugate of claim 1, whereinthe antibody is capable of binding to an antigen expressed on a tumorcell.
 8. The antibody-drug conjugate of claim 1, wherein the drugmolecule comprises an iron complexing moiety that binds to the ferriciron.
 9. The antibody-drug conjugate of claim 1, wherein the drugmolecule is coupled to an iron complexing moiety which binds to theferric iron.
 10. The antibody-drug conjugate of claim 8, or wherein saidiron complexing moiety is selected from the group consisting of ahydroxamate moiety, a catecholate moiety and a carboxylate moiety. 11.The antibody-drug conjugate of claim 1, having the structure:Ab[-L1-Me (-L2-D)_(n)]_(m), wherein Ab is the antibody, L1 is a firstlinker, Me is the ferric iron, L2 is a second linker or absent, D is thedrug molecule, m ranges from 1 to 10, and n ranges from 1 to
 3. 12. Theantibody-drug conjugate of claim 1, wherein the drug molecule isreleased from the ferric iron if the ferric iron is reduced.
 13. Theantibody-drug conjugate of claim 1, wherein the drug molecule isreleased from the ferric iron at a pH of less than
 5. 14. Theantibody-drug conjugate of claim 1, which is stable at pH
 8. 15. Theantibody-drug conjugate of claim 1, which is stable at pH
 5. 16. Apharmaceutical composition comprising the antibody-drug conjugate ofclaim
 1. 17. A method of treating disease in a patient in need thereof,said method comprising the step of administering to said patient aneffective amount of the antibody-drug conjugate of claim 1, or apharmaceutical composition comprising said antibody-drug conjugate. 18.The method of claim 17, wherein said disease is cancer.
 19. A method ofpreventing or reducing the release of a drug molecule from anantibody-drug conjugate in blood, said method comprising the step ofcomplexing a ferric iron with said antibody and said drug molecule toform said antibody-drug conjugate.
 20. The method of claim 19, whereinthe drug molecule is released from the antibody-drug conjugate in theendosomal compartment of a cell in said patient.
 21. The method of claim19, wherein the antibody-drug conjugate is comprised of an antibody, atleast one ferric iron bound to the antibody, and at least one drugmolecule hound to the ferric iron.
 22. (canceled)
 23. (canceled) 24.(canceled)